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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 3, 2003 as doi:10.1096/fj.02-0749fje. |
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* Department of Microbiology and Immunology and
Department of Physiology, University of Arizona, Tucson, Arizona, USA
3Correspondence: University of Arizona, Department of Microbiology and Immunology, 1501 N Campbell, PO Box 245049, Tucson, AZ 85724, USA. E-mail: deluca{at}u.arizona.edu
SPECIFIC AIMS
A major point of contention regarding the well-documented decrease in immune function of animals or astronauts during spaceflight is the potential involvement of stress hormones as opposed to a direct effect of microgravity. The primary aim of this work was to examine the effects of microgravity and vector-averaged gravity, the latter produced by rotation on a clinostat, on the development of T cells in a fetal thymus organ culture (FTOC) system free of stress hormones.
PRINCIPAL FINDINGS
1. Flight rat bone marrow (BM) cells exhibit impaired ability to produce T cells in FTOC
To examine how immune system function is compromised in microgravity, we used an organ culture system that allows for the normal development of rat T cells from hematopoietic stem cell (HSC) progenitors. In collaboration with Drs. Bruce McNaughton and Carol Barnes (Department of Psychology; University of Arizona), we obtained BM and spleen tissue from adult rats flown aboard the STS-90 Neurolab space shuttle mission for 15 days, 21 h, and 50 min, and homologous tissues from their synchronous ground control. Flight rat #1 and the ground control rat were killed at 72 h postlanding and flight rat #2 was killed at 96 h postlanding. BM cell suspensions from the rats’ femurs were prepared and introduced at varying densities into FTOC composed of an equal number of C57BL/6 and NOD scid/scid thymus lobes. After twelve 12 days of culture, there was a decrease in the percentage of CD4 single positive (SP) T cells and CD8 SP T cells in at least one of the concentrations of donor BM used. However, the ability of the BM from both flight rats to produce CD4 T cells was more compromised, compared with the CD8 T cells. The total recovery of double positive (DP) and SP T cells was also decreased in both flight rats. Thus, the ability of flight rat BM to produce T cell precursors was compromised, or the cells were produced but could not migrate to the fetal thymus and differentiate there. Flight rat #2 exhibited growth closer to that of the ground control when compared with flight rat #1, suggesting that the extra 24 h on the ground before BM harvest may have allowed for some recovery in T cell precursor activity.
2. Clinorotation exposure blocks T cell development with the greatest effect occurring in the early stages of organ culture
Tissues from flight rats experienced not only the microgravity of space but also various stresses associated with entry and exit from orbital flight. To differentiate the effects of steroid-associated stress from those of microgravity, we exposed C57BL/6 FTOC (from ground-based mice) to a simulated microgravitational environment produced by averaging the gravity vector (Fig. 1
a, b). This system uses fetal thymus lobes derived from C57BL/6 mice and allows for the study of immune cell development in vitro and away from any outside influences, including stress hormones produced during flight. The percentage of CD4 SP, CD8 SP, and DP T cells produced by 14 day gestation C57BL/6 fetal thymus lobes was reduced after 12 days of clinorotation at 9 ± 0.5 RPM compared with both stationary and motional controls (Fig. 1c, e
). In contrast, the percentage of CD4-CD8- double negative (DN) cells increased. The total cell recovery was also much lower for clinorotated FTOC than it was for the controls. The few cells retrieved were either DN cells or immature single positive cells (ISP, as defined in Fig. 1d
as CD4lo or CD8lo) and shown for frequency and total cell recovery in Fig. 1e
. The frequencies and total recoveries of CD4hi and CD8hi mature SP (MSP) were also considerably lower in the clinorotated FTOC compared with the controls (Fig. 1e
). The frequencies of CD4 ISP and CD8 ISP, with respect to their progeny DP and MSP populations, actually increased compared with the controls, although the total recovery of these cells was decreased due to the overall loss of cell production in the clinorotated cultures. These data suggest that CD4 ISP and CD8 ISP make up a larger proportion of SP T cells in clinorotated cultures. When examining the CD4:CD8 ratio, the clinostat-derived data are also similar to the data obtained from the two flight rat chimeras in that both the vector-averaged gravity and the flight rat-derived cultures exhibited a decrease in production of CD4 T cells relative to CD8 T cells (mean CD4:CD8 ratio for all doses of flight rat precursors used = 0.53 ± 0.21 for flight rat #1, 0.82 ± 0.38 for flight rat #2, and 0.92 ± 0.38 for the synchronous ground control rat; the CD4:CD8 ratio for the clinostat cultures = 0.22 ± 0.06, motional control = 0.39 ± 0.06, and stationary control = 0.39 ± 0.08), suggesting that production of CD4 SP T cells is more acutely affected than the production of CD8 SP T cells.
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To analyze the effects of clinorotation on the different types of T cells that are produced at different stages of FTOC, we examined the approximate time frames during which clinorotation affected the cultures. Experiments were performed in which cultures were clinorotated from day 0 to day 3 and harvested on day 3 or clinorotated from day 3 to day 6 and harvested on day 6. The 0 to 3 day exposure data show that only the immediate precursors of MSP T cells (i.e., the DP T cells) were affected during the 0-–3 days of clinorotation, both in frequency and in total recovery (Fig. 2
a, b). In addition, a similar experiment where the cultures were clinorotated from day 0 to day 3 and harvested on day 12 yielded similar findings. Thus, the overall result was the loss of total DP cell production and an increase in the frequency of ISP cells. H&E stained tissue sections prepared from the FTOC in these experiments exhibited a significant alteration in the structure of the denser staining cortical region and a marked reduction in overall thymus size, consistent with the loss of DP cells (Fig. 2c
). DN cell production was also slightly decreased when these cultures were clinorotated from 0–3 days (Fig. 2a
), suggesting that precursors to ISP may also have been affected. Therefore, we examined the production of pro- and pre-T cells in 2–-3 day clinorotated FTOC by staining for CD4-CD8– "triple negative" [TN] cells, which were also analyzed for CD44 and CD25 expression. The frequency and total cell production of all of the TN pro-T and pre-T cell intermediates were unchanged in clinorotated FTOC. Therefore, the block in T cell development appears to occur at the transition between the late pre-T cell and DP T cell stages (Fig. 3
).
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CONCLUSIONS AND SIGNIFICANCE
Our study provides a demonstration of the importance of gravity for the development of T cells in organ culture. First, we were able to show the effects of spaceflight on the ability of rat BM-derived hematopoietic progenitors to populate murine thymus and differentiate into mature SP T cells. Next, we were able to show that exposure exclusively to vector-averaged gravity, in a system free of stress hormones, significantly affected T cell development in a manner similar to that of the cultures derived from rats exposed to spaceflight. This result suggests that the changes in T cell development in space can be caused by microgravity and are not due solely to stress hormones. Furthermore, the clinostat had its most potent effect on 12 day FTOC in the first 3 days of culture, prior to conventional 
ß TCR formation, as revealed by the significant loss of DP and SP T cells. The lack of change noted in the production of all TN subpopulations, as well as the increase in the frequency of ISP T cells, suggests that the block in T cell development occurs after the pre-T cell stage and is consistent with the increase in ISP cells. We believe that ß-selection is occurring normally since there is no block at the CD44-CD25+ stage. However, the lack of transition from the ISP to the DP stage suggests that a defect of the sequelae of signaling via the ß chain of the pre-TCR (or other receptors, such as acetylcholine receptors) is the means by which T cell development is blocked in clinorotation. This may be due to the altered 3-dimensional structure of the thymus and/or the inability of receptors on developing thymocytes to form multifocal synapses with surrounding thymic epithelial cells.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0749fje; doi: 10.1096/fj.02-0749fje 
2 Current address; R. W. Johnson PRD, La Jolla, CA 92121, USA.
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1 mm away from the axis of rotation. Taking into consideration the close proximity of the lobes to the axis of rotation and the low rate of rotation, the resultant centrifugal force (on the order of 10–4 g) makes a negligible contribution in counteracting the vector-averaged gravity conditions. c) Depicted is a representative flow cytometric profile from a lymphoid gate of FTOC exposed to clinorotation for all 12 days of culture, a motional control, and a stationary control culture. d) Histograms illustrating the intensity (background, low and high) of CD4-TC and CD8-FITC staining on the SP populations and e) cell yields per thymus lobe for DN, CD8lo and CD4lo ISPs, DP, CD8hi, and CD4hi MSPs from 14 day gestation C57BL/6 FTOC that had been cultured for 12 days. Below the yields are tables showing the frequencies of each phenotype. The clinostat and motional control were both rotated at 9 ± 0.5 RPM for the duration of the culture. Data represent the mean ± standard error of the mean (SEM) of the indicated number of determinations. P 
